Hydroxycholesterol immunoassay

Abstract

Provided is an antibody composition comprising antibodies that specifically bind to 22-hydroxycholesterol, 24S-hydroxycholesterol, 25-hydroxycholesterol, 26-hydroxycholesterol or 27-hydroxycholesterol. Also provided is a method of making antibodies that specifically bind to 22-hydroxycholesterol, 24S-hydroxycholesterol, 25-hydroxycholesterol, 26-hydroxycholesterol or 27-hydroxycholesterol is provided. Further provided is a method of assaying for 22-hydroxycholesterol, 24S-hydroxycholesterol, 25-hydroxycholesterol, 26-hydroxycholesterol or 27-hydroxycholesterol is provided. Still further provided is a kit for detecting 22-hydroxycholesterol, 24S-hydroxycholesterol, 25-hydroxycholesterol, 26-hydroxycholesterol or 27-hydroxycholesterol.

What is claimed is: 
     
       1. A method of assaying for 24(S)-hydroxycholesterol in a fluid or tissue sample from a mammal, the method comprising
 combining the sample with antibodies that specifically bind to 24(S)-hydroxycholesterol, 
 wherein the antibodies are generated by (a) preparing a 24(S)-hydroxycholesterol immunogen by conjugating 3-O-succinoyl-24(S)-hydroxycholesterol to a carrier protein for immunization of a rabbit and the carrier protein is attached through a linkage at the 3-position; (b) immunizing the rabbit with the 24(S)-hydroxycholesterol immunogen under conditions such that the immune system of the rabbit makes antibodies to 24(S)-hydroxycholesterol; and (c) collecting the anti-24(S)-hydroxycholesterol polyclonal antibodies from the rabbit; and 
 detecting whether the antibodies specifically bind to 24(S)-hydroxycholesterol from the sample; 
 
       wherein specific antibody binding to 24(S)-hydroxycholesterol from the sample indicates that 24(S)-hydroxycholesterol is present in the sample. 
     
     
       2. The method of  claim 1 , wherein the carrier protein is keyhole limpet hemocyanin, bovine serum albumin, or ovalbumin. 
     
     
       3. The method of  claim 1 , wherein the sample is from cerebrospinal fluid. 
     
     
       4. The method of  claim 1 , wherein the sample is from blood. 
     
     
       5. The method of  claim 1 , wherein the sample is brain tissue. 
     
     
       6. The method of  claim 1 , wherein the sample is from a human that has cognitive impairment, Huntington's disease, Alzheimer's disease, or multiple sclerosis. 
     
     
       7. The method of  claim 1 , performed on a solid phase. 
     
     
       8. The method of  claim 7 , wherein the solid phase is a bead or microplate. 
     
     
       9. The method of  claim 1 , wherein the immunoassay is an enzyme linked immunosorbent assay (ELISA). 
     
     
       10. The method of  claim 9 , wherein the ELISA is an indirect competitive ELISA. 
     
     
       11. The method of  claim 10 , wherein the indirect competitive ELISA comprises binding the anti-24(S)-hydroxycholesterol polyclonal antibodies to a solid phase. 
     
     
       12. The method of  claim 11 , wherein the anti-24(S)-hydroxycholesterol polyclonal antibodies are bound to second antibodies that are directly bound to the solid phase. 
     
     
       13. The method of  claim 1 , comprising a microarray assay. 
     
     
       14. A method of assaying for 24(S)-hydroxycholesterol in a fluid or tissue sample from a mammal; wherein the method employs anti-24(S)-hydroxycholesterol polyclonal antibodies specific to 24(S)-hydroxycholesterol that are obtained by immunizing a rabbit with 3-O-succinoyl-24(S)-hydroxycholesterol conjugated to a carrier protein through a linkage at the 3-position; the method comprising the steps of
 a. noncovalently binding second antibodies to a solid phase; 
 b. adding the anti-24(S)-hydroxycholesterol polyclonal antibodies to the solid phase under conditions such that the second antibodies specifically bind to the anti-24(S)-hydroxycholesterol polyclonal antibodies; 
 c. adding the sample and a biotinylated 24(S)-hydroxycholesterol to the solid phase under conditions such that the biotinylated 24(S)-hydroxycholesterol binds to binding sites on the anti-24(S)-hydroxycholesterol polyclonal antibodies in competition with 24(S)-hydroxycholesterol in the sample; 
 d. adding an enzyme conjugate that is an avidin-enzyme conjugate or a streptavidin-enzyme conjugate to the solid phase under conditions such that the enzyme conjugate specifically binds to the biotinylated 24(S)-hydroxycholesterol that bound in step c; 
 e. adding a substrate to the enzyme in the enzyme conjugate, wherein the substrate is converted to a colored product that absorbs light at a specified wavelength in proportion to the amount of the enzyme bound to the solid phase; and 
 f. measuring the absorbance of light at the specified wavelength, wherein the absorbance is inversely proportional to the amount of 24(S)-hydroxycholesterol present in the sample. 
 
     
     
       15. The method of  claim 14 , wherein, in step c, the sample and the biotinylated 24(S)-hydroxycholesterol are incubated with the solid phase at the same time such that 24(S)-hydroxycholesterol in the sample competes with the biotinylated 24(S)-hydroxycholesterol for anti-24(S)-hydroxycholesterol polyclonal antibody binding sites. 
     
     
       16. The method of  claim 14 , wherein the anti-24(S)-hydroxycholesterol polyclonal antibodies specific to 24(S)-hydroxycholesterol are obtained by immunizing a rabbit with 3-O-succinoyl-24(S)-hydroxycholesterol conjugated to the carrier protein selected from the group consisting of keyhole limpet hemocyanin, bovine serum albumin, and ovalbumin. 
     
     
       17. The method of  claim 14 , wherein the biotinylated 24(S)-hydroxycholesterol has the chemical structure of 
       
         
           
           
               
               
           
         
       
     
     
       18. The method of  claim 14 , wherein the biotinylated 24(S)-hydroxycholesterol has the chemical structure of 
       
         
           
           
               
               
           
         
       
     
     
       19. The method of  claim 14 , wherein the biotinylated 24(S)-hydroxycholesterol is a biotin derivative of 3-O-succinoyl-24(S)-hydroxycholesterol N-hydroxysuccinimide ester having the chemical structure of